Bacillus thuringiensis isolate BTC-18 with broad spectrum activity

ABSTRACT

A broad spectrum  Bacillus thuringiensis  strain, BtC-18, is provided which displays pesticidal activity against nematodes and against insects from the orders Lepidoptera, Diptera and Coleoptera.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.09/003,217, filed Jan. 6, 1998, now U.S. Pat. No. 5,986,177, whichclaims the benefit of Provisional Application Serial No. 60/035,361,filed Jan. 10, 1997, the disclosures of which are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to novel Bacillus thuringiensis strains,to novel toxin genes, to the proteins encoded by the genes, and to theuse of genes and proteins.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a gram-positive soil bacterium characterizedby its ability to produce crystalline inclusions during sporulation. Thecrystalline inclusions can, in some subspecies, account for 20 to 30percent of the dry weight of the sporulated cell and may be composed ofmore than one protein. Crystals are composed primarily of a singlepolypeptide, a protoxin, which may also be a component of the sporecoat. The protoxin genes are located mainly on large plasmids, althoughchromosomally encoded endotoxins have been reported.

The crystal proteins exhibit a highly specific insecticidal activity.Many B. thuringiensis strains with different insect host spectra havebeen identified. They are classified into different serotypes orsubspecies based on their flagellar antigens.

The protoxin does not exhibit its insecticidal activity until after oralintake of the crystalline body. The crystal is dissolved in theintestinal juice of the target insects. In most cases, the actual targetcomponent (toxin) is released from the protoxin as a result ofproteolytic cleavage caused by the action of proteases from thedigestive tract of the insects. The activated toxin interacts with themidgut epithelium cells of susceptible insects.

Electrophysiological and biochemical evidence suggests that the toxinsgenerate pores in the cell membrane, thus disrupting the osmoticbalance. Consequently, the cells swell and lyse. For several B.thuringensis toxins, specific high-affinity binding sites have beendemonstrated to exist on the midgut epithelium of susceptible insects.Nucleotide sequences have been recorded for a large number of B.thuringiensis (Bt) crystal protein genes. Several sequences are nearlyidentical, and have been designated as variations of the same gene. Thecrystal protein (Cry) genes specify a family of related insecticidalproteins. The genes are divided into major classes and subclassescharacterized by both the structural similarities and the insecticidalspectra of the encoded proteins. The classification, explained by Höfteand Whiteley (1989) Microbiol. Rev., 53:242-255, placed the knowninsecticidal crystal proteins into four major classes. The four majorclasses were Lepidoptera-specific (I), Lepidoptera- and Diptera-specific(II), Coleoptera-specific (III), and Diptera-specific (IV) genes.Additional classes have since been added.

The Cry1 genes are undoubtedly the best-studied crystal proteins. TheCry1 proteins are typically produced as 130 to 140 kDa protoxin proteinswhich are proteolitically cleaved to produce active toxin proteins about60 to 70 Kda. The active portion or toxic domain is localized in theN-terminal half of the protoxin. Six groups of Cry1 proteins were knownin 1989 when the Höfte and Whiteley article was published. These groupswere designated IA(a), IA(b), IA(c), IB, IC, and ID. Since 1989,additional proteins have been discovered and classified as Cry1E, Cry1F,Cry1G, Cry1H, and Cry1X.

The spectrum of insecticidal activity of an individual protoxin from Bttends to be quite narrow. That is, a given crystal protein is activeagainst only a few insects. None of the crystal proteins active againstColeopteran larvae such as colorado potato beetle (Leptinotarsadecemlineata) or yellow mealworm (Tenebrio molitor) have demonstratedsignificant effects on members of the genous Diabrotica particularly D.virgifera virgifera, the western corn rootworm (WCRW) or D. longicornisbarberi, the northern corn rootworm.

Insect pests are a major factor in the loss of the world's commerciallyimportant agricultural crops. Broad spectrum chemical pesticides havebeen used extensively to control or eradicate pests of agriculturalimportance. However, there is substantial interest in developingeffective alternative pesticides.

Microbial pesticides have played an important role as alternatives tochemical pest control. The most extensively used microbial product isbased on the bacterium Bacillus thuringiensis. However, as noted above,the majority of Bt strains have a narrow range of activity. There istherefore needed microbial strains with a broad range of insecticidalactivity for use as broad spectrum insecticides and as a source foradditional toxin genes and proteins.

SUMMARY OF THE INVENTION

A broad spectrum Bacillus strain is provided. The strain is activeagainst insects from at least the orders Lepidoptera, Diptera, andColeoptera. Additionally, the strain is active against nematodes, androotworms. Disclosed in this invention is the isolation and partialpurification of the crystal protein complex from this strain. Thecrystal protein complex is demonstrated to be active against rootwormsand other pests. Genes, proteins, and their use, as well as the use ofthe strain are provided. The complete nucleotide sequence of a Cry5-likegene herein designated as Cry1I which was isolated from this strain isalso provided.

The methods and compositions of the invention may be used in a varietyof systems for controlling pests, particularly plant pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: FIG. 1A shows an electron micrograph with a lightmicrograph insert; FIG. 1B shows an electron micrograph with thedifferent kinds of crystal shapes as indicated by the arrows or arrowheads. The three types of crystal shapes may correlate with the threekinds of insecticidal activity toward at least three insect orders,namely: Lepidoptera, Diptera and Coleoptera.

FIG. 2: Plasmid profile of BtC-18. Lane a: DNA molecular weightstandards; Lane b: B. thuringiensis subsp. kurstaki; Lane c: Bacillusthuringiensis subsp. aizawia; and, Lane d: strain C-18. BtC-18 also hasdifferent plasmid DNA profiles from the other known B. thuringiensissubspecies, such as subspecies israelensis and tenebrionis, (data notshown).

FIGS. 3A-3C: Protective effect of BtC-18 against the root nematodeMeloidogyne incognita. In panel A: the roots of infected tomato plantwith the nematode (positive control). panel B: the roots of tomatoplants infected with nematode eggs before BtC-18 was applied, and panelC: roots of plant treated first with BtC-18 and then infected withnematode eggs.

FIGS. 4A-4B: PCR product profile of BtC-18. In FIG. 4A: Lane a containsDNA molecular weight standards; Lane b, B. thuringiensis subsp.kurstaki; Lane c, BtC-18; Lane d: B. thuringiensis subsp. israelensis;Lane e, BtC-18. In FIG. 4B: Lane a contains DNA standards; Lane f,Bacillus thuringiensis subsp. tenebrionis; Lane g, BtC-18. The resultsshow that BtC-18 produced the same PCR product profile as B.thuringiensis subsp. kurstaki, but different PCR products with Dipteranand Coleopteran DNA primers.

FIGS. 5A-5E. Nucleotide and amino acid sequence of the Cry1I geneisolated from BtC-18.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for controlling plant pests are provided. Inparticular, novel broad spectrum Bacillus strains having a wide range ofinsecticidal activity are provided. The strains are useful asinsecticidal agents. In addition, the crystal protein complex from oneof these strains has been purified and is shown to have insecticidalproperties. Methods of its purification are discussed and evidence ofits activity against rootworm is disclosed. Also disclosed is the entirenucleotide sequence of a Cry5-like gene herein designated as Cry1I whichwas isolated from this strain.

The Bacillus strain of the invention has a broad spectrum of activity.By broad spectrum it is intended that the strains are active againstinsects from more than one order, preferably against insects fromseveral orders, more preferably active against insects from at leastthree orders. Additionally, the strains are active against noninsectpests. For purposes of the present invention, pests include but are notlimited to insects, fungi, bacteria, nematodes, mites, ticks, and thelike. Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera, Lepidoptera, and Diptera.

In one embodiment, the invention encompasses the Bt isolate known asBtC-18, deposited Dec. 31, 1996 as ATCC Accession No. 55922 (AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.). Proteintoxins, and DNA which encodes the protein toxins are additionallyencompassed. The subject invention also includes variants of the Btisolate which have substantially the same pesticidal properties as theexemplified isolate. These variants include mutants and recombinantisolates. Procedures for making mutants are well known in the art andinclude ultraviolet light and nitrosoguanidine.

The Bacillus thuringiensis isolate C-18 produces more than one kind ofcrystal protein during sporulation. Microscopic examination of thesporulated cells revealed at least three different shapes of crystals:bipyrmidal similar to the lepidopteran-specific B. thuringiensis subsp.kurstaki; circular or irregular as that produced by dipteran-specific B.thuringiensis subsp. israelensis; and rhomboid-shaped similar to thecoleopteran-specific B. thuringiensis subsp. tenebrionis.

The presence of the different types of crystals indicated that thebacterium can kill insects belonging to more than one order of insects.Bioassays confirmed that conclusion. The spore-crystal complex of C-18killed insects from at least three orders, Lepidoptera, Diptera, andColeoptera. Generally, Bt's produce only a single crystal and thereforehave limited insect activity. Of the Bt's which have been reported tokill insects from two orders, the activity is not stable. In contrast,the present Bt has been shown to be highly stable.

Of particular interest is the corn rootworm activity exhibited by theBacillus strains of the invention. Generally, Bt's have little or norootworm activity. In contrast, the present strain exhibits substantialrootworm activity. By substantial activity is intended that the proteinis capable of killing the target insect when present in at leastmicrogram (μg) quantities.

Methods are available in the art for the identification and isolation ofthe protein or proteins associated with insecticidal activity, i.e.,rootworm activity. Generally, proteins can be purified by conventionalchromatography, including gel-filtration, ion-exchange, andimmunoaffinity chromatography, by high-performance liquidchromatography, such as reversed-phase high-performance liquidchromatography, ion-exchange high-performance liquid chromatography,size-exclusion high-performance liquid chromatography, high-performancechromatofocusing and hydrophobic interaction chromatography, etc., byelectrophoretic separation, such as one-dimensional gel electrophoresis,two-dimensional gel electrophoresis, etc. Such methods are known in theart. See for example, Ausubel et al. (1988) Current Protocols inMolecular Biology, Vols. 1 & 2, (eds.) John Wiley & Sons, NY.

Additionally, antibodies can be prepared against substantially purepreparations of the protein. See, for example, Radka et al. (1983) J.Immunol., 128:2804; and Radka et al. (1984) Immunogenetics, 19:63. Anycombination of methods may be utilized to purify protein havingpesticidal properties, particularly rootworm activity. As the protocolis being formulated, insecticidal activity is determined after eachpurification step to assure the presence of the toxin of interest.

Methods are available in the art to assay for insect activity.Generally, the protein is mixed and used in feeding assays. See, forexample Marrone et al. (1985) J. of Economic Entomology, 78:290-293.

Such purification steps will result in a substantially purified proteinfraction. By “substantially purified” or “substantially pure” isintended protein which is substantially free of any compound normallyassociated with the protein in its natural state. “Substantially pure”preparations of protein can be assessed by the absence of otherdetectable protein bands following SDS-PAGE as determined visually or bydensitometry scanning. Alternatively, the absence of otheramino-terminal sequences or N-terminal residues in a purifiedpreparation can indicate the level of purity. Purity can be verified byrechromatography of “pure” preparations showing the absence of otherpeaks by ion exchange, reverse phase or capillary electrophoresis. Theterms “substantially pure” or “substantially purified” are not meant toexclude artificial or synthetic mixtures of the proteins with othercompounds. The terms are also not meant to exclude the presence of minorimpurities which do not interfere with the biological activity of theprotein, and which may be present, for example, due to incompletepurification.

Once purified protein is isolated, the protein, or the polypeptides ofwhich it is comprised, can be characterized and sequenced by standardmethods known in the art. For example, the purified protein, or thepolypeptides of which it is comprised, may be fragmented as withcyanogen bromide, or with proteases such as papain, chymotrypsin,trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem.,257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res.,21:209-215). The resulting peptides are separated, preferably by HPLC,or by resolution of gels and electroblotting onto PVDF membranes, andsubjected to amino acid sequencing. To accomplish this task, thepeptides are preferably analyzed by automated sequencers. It isrecognized that N-terminal, C-terminal, or internal amino acid sequencescan be determined. From the amino acid sequence of the purified protein,a nucleotide sequence can be synthesized which can be used as a probe toaid in the isolation of the gene encoding the pesticidal protein.

Similar protocols can be utilized to isolate nematode active proteins,rootworm active proteins, or proteins having activity to other pests.

It is recognized that the pesticidal proteins may be oligomeric and willvary in molecular weight, number of protomers, component peptides,activity against particular pests, and in other characteristics.However, by the methods set forth herein, proteins active against avariety of insects or pests may be isolated and characterized.

Once the purified protein has been isolated and characterized it isrecognized that it may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the pesticidal proteins can be prepared bymutations in the DNA. Such variants will possess the desired pesticidalactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary rnRNA structure. See, EP Patent Application Publication No.75,444.

In this manner, the present invention encompasses the active rootwormproteins, and other insecticidal and pesticidal proteins, as well ascomponents and fragments thereof. That is, it is recognized thatcomponent protomers, polypeptides or fragments of the proteins may beproduced which retain pesticidal activity. These fragments includetruncated sequences, as well as N-terminal, C-terminal, internal andinternally deleted amino acid sequences of the proteins.

Most deletions, insertions, and substitutions of the protein sequenceare not expected to produce radical changes in the characteristics ofthe pesticidal protein. However, when it is difficult to predict theexact effect of the substitution, deletion, or insertion in advance ofdoing so, one skilled in the art will appreciate that the effect will beevaluated by routine screening assays.

The proteins or other component polypeptides described herein may beused alone or in combination. That is, several proteins may be used tocontrol different insect pests.

The pesticidal proteins of the invention can be used in combination withBt toxins or other insecticidal proteins to increase insect targetrange. Other insecticidal principles include protease inhibitors (bothserine and cysteine types), lectins, α-amylase and peroxidase. Thisco-expression of more than one insecticidal principle in the sametransgenic plant can be achieved by genetically engineering a plant tocontain and express all the genes necessary. Alternatively, separateplants can be transformed with different components. By crossing theplants, progeny are obtained which express all of the genes of interest.

It is recognized that there are alternative methods available to obtainthe nucleotide and amino acid sequences of the present proteins. Forexample, to obtain the nucleotide sequence encoding the pesticidalprotein, cosmid clones, which express the pesticidal protein, can beisolated from a genomic library. From larger active cosmid clones,smaller subclones can be made and tested for activity. In this manner,clones which express an active pesticidal protein can be sequenced todetermine the nucleotide sequence of the gene. Then, an amino acidsequence can be deduced for the protein. For general molecular methods,see, for example, Molecular Cloning, A Laboratory Manual, SecondEdition, Vols. 1-3, Sambrook et al. (eds.) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), and the references citedtherein.

The present invention also encompasses nucleotide sequences from otherBacillus strains and organisms other than Bacillus, where the nucleotidesequences are isolatable by hybridization with the Bacillus nucleotidesequences of the invention. Proteins encoded by such nucleotidesequences can be tested for pesticidal activity. The invention alsoencompasses the proteins encoded by the nucleotide sequences.Furthermore, the invention encompasses proteins obtained from organismsother than Bacillus wherein the protein cross-reacts with antibodiesraised against the proteins of the invention. Again the isolatedproteins can be assayed for pesticidal activity by the methods disclosedherein or others well-known in the art.

In this manner, the insecticidal genes of the present invention includethose coding for proteins homologous to, and having essentially the samebiological properties as, the insecticidal genes disclosed herein, andparticularly the rootworm active gene. This definition is intended toencompass natural allelic variations in the genes. Cloned genes of thepresent invention can be of other species of origin. Thus, DNAs whichhybridize to the present insecticidal genes are also an aspect of thisinvention. Conditions which will permit other DNAs to hybridize to theDNA disclosed herein can be determined in accordance with knowntechniques. For example, hybridization of such sequences may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% Formamide with 5× Denhardt's solution, 0.5% SDS and 1×SSPE at37° C.; conditions represented by a wash stringency of 40-45% Formamidewith 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; andconditions represented by a wash stringency of 50% Formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively, toDNA encoding the insecticidal genes disclosed herein in a standardhybridization assay. See J. Sambrook et al., Molecular Cloning, ALaboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory)). Ingeneral, sequences which code for insecticidal protein and hybridize tothe insecticidal gene disclosed herein will be at least 75% homologous,85% homologous, and even 95% homologous or more with the sequences.Further, DNAs which code for insecticidal proteins, or sequences whichcode for an insecticidal protein coded for by a sequence whichhybridizes to the DNAs which code for insecticidal genes disclosedherein, but which differ in codon sequence from these due to thedegeneracy of the genetic code, are also an aspect of this invention.The degeneracy of the genetic code, which allows different nucleic acidsequences to code for the same protein or peptide, is well known in theliterature. See, e.g., U.S. Pat. No. 4,757,006 to Toole et al. at Col.2, Table 1.

The hybridization probes may be cDNA fragments or oligonucleotides, andmay be labeled with a detectable group as discussed hereinbelow. Pairsof probes which will serve as PCR primers for the insecticidal gene or aprotein thereof may be used in accordance with the process described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

Once the nucleotide sequences encoding the pesticidal proteins of theinvention have been isolated, they can be manipulated and used toexpress the protein in a variety of hosts including other organisms,including microorganisms and plants.

The pesticidal genes of the invention can be optimized for enhancedexpression in plants. See, for example, EPA 0359472; EPA 0385962; WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328;and Murray et al. (1989) Nucleic Acids Research 17:477-498. In thismanner, the genes can be synthesized utilizing plant preferred codons.That is the preferred codon for a particular host is the single codonwhich most frequently encodes that amino acid in that host. The maizepreferred codon, for example, for a particular amino acid may be derivedfrom known gene sequences from maize. Maize codon usage for 28 genesfrom maize plants is found in Murray et al. (1989) Nucleic AcidsResearch, 17:477-498, the disclosure of which is incorporated herein byreference. Synthetic genes can also be made based on the distribution ofcodons a particular host uses for a particular amino acid.

In this manner, the nucleotide sequences can be optimized for expressionin any plant. It is recognized that all or any part of the gene sequencemay be optimized or synthetic. That is, synthetic or partially optimizedsequences may also be used.

In like manner, the nucleotide sequences can be optimized for expressionin any microorganism. For Bacillus preferred codon usage, see, forexample U.S. Pat. No. 5,024,837 and Johansen et al. (1988) Gene,65:293-304.

Methodologies for the construction of plant expression cassettes as wellas the introduction of foreign DNA into plants are described in the art.Such expression cassettes may include promoters, terminators, enhancers,leader sequences, introns and other regulatory sequences operably linkedto the pesticidal protein coding sequence.

Methodologies for the construction of plant expression cassettes aredescribed in the art. The construct may include any necessary regulatorssuch as terminators, (Guerineau et al. (1991) Mol. Gen. Genet.,226:141-144; Proudfoot (1991) Cell, 64:671-674; Sanfacon et al. (1991)Genes Dev., 5:141-149; Mogen et al. (1990) Plant Cell, 2:1261-1272;Munroe et al. (1990) Gene, 91:151-158; Ballas et al. (1989) NucleicAcids Res., 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res.,15:9627-9639); plant translational consensus sequences (Joshi, C. P.(1987) Nucleic Acids Research, 15:6643-6653), enhancers, introns(Luehrsen and Walbot (1991) Mol. Gen. Genet., 225:81-93) and the like,operably linked to the nucleotide sequence. It may be beneficial toinclude 5′ leader sequences in the expression cassette construct. Suchleader sequences can act to enhance translation. See, for example,(Elroy-Stein et al. (1989) PNAS USA, 86:6126-6130), Allison et al.(1986), Macejak and Sarnow (1991) Nature, 353:90-94, Jobling and Gehrke(1987) Nature, 325:622-625, Gallie et al. (1989) Molecular Biology ofRNA, pp. 237-256, Lommel et al. (1991) Virology, 81:382-385, andDella-Cioppa et al. (1987) Plant Physiology, 84:965-968.

It is further recognized that the components of the expression cassettemay be modified to increase expression. For example, truncatedsequences, nucleotide substitutions or other modifications may beemployed. See, for example Perlak et al. (1991) Proc. Natl. Acad. Sci.USA, 88:3324-3328; Murray et al. (1989) Nucleic Acids Research,17:477-498; and WO 91/16432.

For tissue specific expression, the nucleotide sequences of theinvention can be operably linked to tissue specific promoters.

Methods are available in the art for the introduction and stableincorporation of the expression cassettes into plants. Suitable methodsof transforming plant cells include microinjection (Crossway et al.(1986) Biotechniques, 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA, 83:5602-5606, Agrobacterium mediatedtransformation (Hinchee et al. (1988) Biotechnology, 6:915-921), directgene transfer (Paszkowski et al. (1984) EMBO J., 3:2717-2722), andballistic particle acceleration (see, for example, Sanford et al., U.S.Pat. No. 4,945,050; WO91/10725 and McCabe et al. (1988) Biotechnology,6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet.,22:421-477; Sanford et al. (1987) Particulate Science and Technology,5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology, 6:923-926 (soybean);Datta et al. (1990) Biotechnology, 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA, 85:4305-4309 (maize); Klein et al. (1988)Biotechnology, 6:559-563 (maize); WO91/10725 (maize); Klein et al.(1988) Plant Physiol., 91:440-444 (maize); Fromm et al. (1990)Biotechnology, 8:833-839; and Gordon-Kamm et al. (1990) Plant Cell,2:603-618 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature(London), 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci.USA, 84:5345-5349 (Liliaccae); De Wet et al. (1985) In The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman et al. pp. 197-209.Longman, N.Y. (pollen); Kaeppler et al. (1990) Plant Cell Reports,9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet., 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell,4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports,12:250-255 and Christou and Ford (1995) Annals of Botany, 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology, 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

Alternatively, the plant plastid can be transformed directly. Stabletransformation of plastids have been reported in higher plants, see, forexample, Svab et al. (1990) Proc. Nat'l. Acad. Sci. USA, 87:8526-8530;Svab & Maliga (1993) Proc. Nat'l Acad. Sci. USA, 90:913-917; Staub &Maliga (1993) EMBO J., 12:601-606. The method relies on particular gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by trans-activation of asilent plastid-borne transgene by tissue-specific expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci., USA,91:7301-7305.

The cells which have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports, 5:81-84 (1986). These plants may then begrown, and either pollinated with the same transformed strain ordifferent strains, and the resulting hybrid having the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that the subject phenotypic characteristic is stablymaintained and inherited and then seeds harvested to ensure the desiredphenotype or other property has been achieved.

The Bacillus strains of the invention may be used for protectingagricultural crops and products from pests. Alternatively, a geneencoding the pesticide may be introduced via a suitable vector into amicrobial host, and said host applied to the environment or plants oranimals. Microorganism hosts may be selected which are known to occupythe “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplana) of one or more crops of interest. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

A number of methods are available for introducing a gene expressing thepesticidal protein into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. For example,expression cassettes can be constructed which include the DNA constructsof interest operably linked with the transcriptional and translationalregulatory signals for expression of the DNA constructs, and a DNAsequence homologous with a sequence in the host organism, wherebyintegration will occur, and/or a replication system which is functionalin the host, whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include but are notlimited to promoter, transcriptional initiation start site, operators,activators, enhancers, other regulatory elements, ribosomal bindingsites, an initiation codon, termination signals, and the like. See, forexample, U.S. Pat. No. 5,039,523; U.S. Pat. No. 4,853,331; EPO0480762A2; Sambrook et al. supra; Molecular Cloning, a LaboratoryManual, Maniatis et al. (eds) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982); Advanced Bacterial Genetics, Davis et al. (eds.)Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980); and thereferences cited therein.

General methods for employing the strains of the invention in pesticidecontrol or in engineering other organisms as pesticidal agents are knownin the art. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains of the invention or the microorganisms which havebeen genetically altered to contain the pesticidal gene and protein maybe used for protecting agricultural crops and products from pests. Inone aspect of the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticides are produced by introducing a heterologousgene into a cellular host. Expression of the heterologous gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. These cells are then treated under conditions thatprolong the activity of the toxin produced in the cell when the cell isapplied to the environment of target pest(s). The resulting productretains the toxicity of the toxin. These naturally encapsulatedpesticides may then be formulated in accordance with conventionaltechniques for application to the environment hosting a target pest,e.g., soil, water, and foliage of plants. See, for example EPA 0192319,and the references cited therein.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be both fertilizers or micronutrient donors or otherpreparations that influence plant growth. They can also be selectiveherbicides, insecticides, fungicides, bacteriocides, nematocides,mollusocides or mixtures of several of these preparations, if desired,together with further agriculturally acceptable carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g. natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders or fertilizers.

Preferred methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention whichcontains at least one of the pesticidal proteins produced by thebacterial strains of the present invention are leaf application, seedcoating and soil application. The number of applications and the rate ofapplication depend on the intensity of infestation by the correspondingpest.

The following experiments are offered by way of illustration and not byway of limitation.

EXPERIMENTAL Introduction

Bacillus thuringiensis (Bt) strain C-18 is a gram-positive sporeformingbacterium that produces parasporal crystals which have multiple toxicityagainst three orders of insects: Lepidoptera, Coleoptera and Diptera aswell as to nematodes. C-18 is unique because of its capacity to killsuch a wide range of agriculturally and biomedically important pests. Noother Bt strain or isolate has been reported to have such a wide hostrange. The vast majority of Bt's can kill insects belonging to only oneorder of insects.

Isolation

C-18 was isolated in Egypt from dead pink bollworm larvae harvested fromcotton bolls grown in cotton fields in Egypt. Standard bacteriologicalprocedures were used to isolate gram-positive, spore forming bacteria.The larvae were washed twice with sterile deionized water, transferredto fresh sterile water (1 ml), macerated with a sterile glass-rod beforebeing subjected to a heat treatment (5 min. at 100° C.). The heattreatment killed all the vegetative and non-sporulating microbes. 100 μlsamples were then streaked onto LB-agar plates and incubated at 30° C.overnight. Individual colonies were then picked, streaked onto freshsporulating medium and incubated at 30° C. for 48 hours.

The resulting colonies were stained with endospore stain and examinedmicroscopically. All standard bacteriological techniques includingphysiological and biochemical reactions were employed to determine theidentity of the isolates and only those which matched Bacillusthuringiensis (Bt) were subjected to detailed analysis and evaluation.Among the isolates selected for determining insecticidal and nematocidalactivities was C-18.

A unique aspect of the isolate C-18 was its ability to produce more thanone kind of protein crystal during sporulation. Microscopic examinationof the stained sporulated cells revealed at least three different shapesof crystals: bipyrimidial similar to the lepidopteran-specific B.thuringiensis subsp. kurstaki; circular or irregular as that produced byB. thuringiensis subsp. israelensis; and, rhomboid-shaped similar to theB. thuringiensis subsp. tenebrionis (FIGS. 1 A&B).

Insect & Nematode Bioassays

The insecticidal activity of C-18 was measured using bioassays involvinga variety of insects that represent the Lepidoptera, Coleoptera andDiptera orders of insects. Initial insect bioassays were performed withwhole bacterial cells and included the raspberry silkworm Philosamiaricini (Lepidopteran), the mosquito Culex pipiens (Dipteran), and theflour beetle Tribollium spp. (Coleopteran). Nematode bioassays were donewith the tomato nematode Meloidogyne incognita. Positive results fromthese bioassays Table 1 prompted a more comprehensive analysis of theinsecticidal and nematocidal activities of C-18 parasporal crystals. Ascan be seen in Table 1, C-18 is effective against a broad spectrum ofinsects. Also parasporal crystals purified from C-18 exhibited the samenematocidal activity as the whole organism (FIG. 1).

A more comprehensive bioassay system using larger numbers of insectrepresenting the three orders mentioned above was then performed.

In Table 1, bioassays were divided into three groups. Group I containsthe results of the lepidopteran insects (cotton leafworm Spodopteralittoralis, cotton bollworm Pectinophora gossypiella, tobacco hornwormManduca sexta, raspberry silk worm Philosamia ricini, and a corn borerSesamia cratica). Group II includes dipteran insects (mosquitoes Culexpipiens, Aedes aegypti, Aedes albopictus, Aedes triseriatus, and a bluetongue virus vector: Culicoides variipennis). Group III represents thecoleopteran insects (flour beetle Tribollium spp., Colorado potatobeetle Leptinotarsa decemlineata, western corn rootworm Diaborticavirgifera and southern corn rootworm D. undecimpuncata howardi).

The bacterium was also tested against the nematode Melodogyne incognita(FIG. 3).

TABLE 1 Pesticidal Activities of an Egyptian Isolate Bacillusthuringiensis (BtC-18) INSECT BtC-18 BTK BTI BTT Group I:Lepidopteran-insects Spodoptera littotalis ¹ 10.00 40.00 0.0 0.0Pectinophera gossypiella ¹ 0.45 40.00 0.0 0.0 Manduca sexta ¹ 0.45 7.000.0 0.0 Philosamia ricini ¹ 0.43 3.00 0.0 0.0 Sessemia cratia ¹ 20.0030.00 0.0 0.0 Group II: Dipteran-insects Culex pipiens ² 7.00 0.0 5.500.0 Aedes aegypti ² 200.00 0.0 40.00 0.0 Aedes albopictus ² 36.00 0.0 nd0.0 Aedes triseriatus ² 360.00 0.0 nd 0.0 Culicoides variipennis ² 18.000.0 NO 0.0 Group III: Coleopteran-insects Tribollium sp.³ 35.00 0.0 0.030.00 Colorado Potato Beetle¹ 50.00 0.0 0.0 25.00 Western Corn Rootworm¹350.00 0.0 0.0 8,000.00 Southern Corn Rootworm¹ 350.00 0.0 0.0 10,000.00¹The LC50 is expressed in ng/cm.² ²The LC50 is expressed in ng/ml. ³TheLC50 is expressed in ng/g. *BtC-18 also exhibited toxic activity againstNematodes.

Microscopic Examination of C-18

C-18 produces at least three morphological types of parasporal crystals(FIG. 1). The three types or shapes are: (i) bipyrimidal—characteristicof those crystals that are toxic to lepidopterans, (ii) rounded,amorphous clusters—characteristic of dipteran-specific crystals, and(iii) rhomboid—characteristic of coleopteran-specific crystals. Presenceof these three morphological crystal types correlate with the threekinds of insecticidal activities normally associated singularly with allother Bt's described in the scientific literature.

Profile of Proteins Extracted from Vegetative & Sporulating Cells ofC-18

The molecular characterization of BtC-18 was determined at the proteinand the gene levels. Bacillus thuringiensis has two phases of growth,the vegetative phase and the sporulation phase. The bacterium produces aspecific protein pool during each phase of growth. The proteins are theexpression products of active genes at the specific stage ofdevelopment. These proteins can be separated and visualized on a sodiumdedocylsulphate-polyacrylamide gel by electrophoresis (SDS-PAGE).

Proteins of vegetative and sporulating cells of the various strains ofBt described in the literature have common and characteristic bandingpatterns when analyzed by SDS-polyacrylamide gel electrophoresis. Todetermine whether C-18 has a distinctive protein profile of its own,proteins extracted from both vegetative and sporulating cells wereexamined by this technique and compared with Bt subspecies kurstaki(lepidopteran-specific), israeliensis (dipteran-specific), andtenebrionsis (coleopteran-specific). C-18 does, indeed, have distinctiveprotein profiles when compared with several other commonly knownsubspecies of Bt.

Immunochemical Staining of Crystal Proteins

Western analysis of purified crystal proteins from C-18 was performedand proteins identified by this technique were compared with the samethree subspecies of Bt mentioned above. Although there are somesimilarities among the four organisms, C-18 does exhibit a distinctiveand characteristic crystal protein profile.

Plasmid DNA Profiles

Genes responsible for encoding insecticidal proteins that constituteparasporal crystals of Bt usually are associated with plasmid DNAalthough there is evidence that such genes are chromosomally linked aswell. The plasmids purified from C-18 are displayed in FIG. 2. Theplasmid profile of C-18 is distinctive when compared to the profiles ofthe same three subspecies of Bt indicated above.

This bacterium contains a large number of plasmid DNA molecules. Themajority of the toxin genes have been reported to be carried on one ofthe large plasmids, however, few have been reported on the chromosome ofsome Bts. The plasmid profile of C-18 has been used as a tool todifferentiate between bacterial species. The plasmid profile of theBtC-18 was found to be different from the other subspecies of B.thuringiensis tested as indicated in FIG. 2.

Polymerase Chain Reaction (PCR)

To determine the existence of genes (Cry genes) in C-18 that may encodedifferent insecticidal proteins (Cry proteins), specific DNA primerswere constructed and used in PCR analysis of C-18 genomic DNA. Theprimers were custom designed against conserved regions in the genomes ofthe same subspecies used above. Amplification of various DNA fragmentsrevealed that C-18 contains similar Cry1 (Lepidopteran-specific) genesas Bt subsp. kurstaki but different Cry genes that those of Btsubspecies israelensis and tenebrionis (FIG. 4).

The proof of the existence or absence of the three genes encoding thethree crystals produced by BtC-18 was proven by the use of specific DNAprimers (three sets, each consisting of two pairs specific for one gene)designed against conserved regions in the genomes of B. thuringiensissubsp. kurstaki (Lep1A, Lep1B, Lep2A, and Lep2B), B. thuringiensissubsp. israelensis (Dip1A, Dip1B, Dip2A, and Dip2B) and B. thuringiensissubsp. tenebrionis (Col1A, Col1B, Col2A, and Col2B).

The primers were used in the polymerase chain reaction (PCR) to givespecific product profiles. The two pairs of Lep primers amplify a 0.49kb and a 0.908 kb DNA fragment. The Dip primers amplify a 0.797 kb and1.290 kb DNA fragment. The Col primers amplify a 0.649 kb and 1.060 kbDNA fragments (FIG. 4).

Western blot analysis of the crystal proteins produced by BtC-18 confirmthat the crystal proteins of BtC-18 are different from the subspeciesisraelensis and tenebrionis.

Purification of the C18 Crystal Protein Complex and InsecticidalProperties

The BtC18 crystal complex was purified from the sporulated culture brothby Renographin gradient from the methods discussed in Lee et al. (1995)Biochem. Biophys. Res. Comm. 126: 953-960. The purified crystals werewashed several times with de-ionized water to get rid of anycontaminants. The crystal complex was subjected to differentialsolublization at various pH's according to the methods of Dai and Gill(1993) Insect Biochem. Molec. Biol. 23:273-283 and Hofte et al. (1986)Eur. J. Biochem. 161:273-280 and fractions (F1, F2, F3, F4, F5, and F6)obtained from this procedure were bioassayed against corn rootworms(southern and western) as representative of coleopteran insects andtobacco hornworm as lepidopteran insects. All fractions killed bothrepresentative insects, but with varying degrees. Fractions F2, F4, andF6 killed rootworms very efficiently. 5 μg/cm² of the fractions killed80-90% of the tested insects. 100% kill was recorded at 10 μg/cm². Allfractions killed the tobacco hornworm albeit with variable efficiencies.

Binding of BtC18 Toxins to Specific Brush Border Membrane Proteins Fromthe Midgut of Different Insects

Brush border membrane vesicles (BBMV) were prepared from: corn rootworms(WCRW and SCRW), tobacco hornworm (MS), and European corn borer (ECB)according to methods used by Wolfersberger et al. (1987) Comp. Biochem.Physiol. 86A:301-308. Specific amounts (20-MS, 50-ECB, and 120-W/SCRWμg/lane) of BBMV's were loaded and separated by SDS-PAGE. The separatedproteins were electro-blotted onto PVDF nylon membranes and were reactedwith 125 I labeled fractions (F2, F4, F5, and F6) from BtC18, which bindto specific receptors from the BBMV. The radioactive membranes are thenexposed on film. Where there's a band there is an interaction with thespecific receptor. Evident in this analysis is the broad basedinteraction of F6 radiolabelled proteins with the various brush bordermembrane proteins extracted from these pests (Table 2). Based on thisobservation, F6 appears to have the largest range of activity.

TABLE 2 BtC18 Protein BBMV Binding Protein (kDa) fraction MS ECB SCRWWCRW F2 210  nd nd nd F4 210  nd nd nd 84 nd nd nd 45 nd nd nd F5 210 nd nd nd 120  nd nd nd 60 nd nd nd 55 nd nd nd F6 210  210 210 nd 120  84 nd 180 60 nd 150 150 45 nd  50  50 40 nd  42  42

Identification of Cry Genes

DNA primers available in the scientific literature enabled theidentification of the types of genes BtC-18 isolate. Some of the primerswere designed to differentiate between the members of the same family ofgenes such as Cry1 family, Cry2 genes, Cry3 genes, Cry4 genes and Cry5gene. These primers were used in PCR analysis to determine the numberand kinds of the different genes present in the isolate BtC-18. Severalgenes were determined to be present in BtC-18. The identified genes wereCry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry2A, Cry2B, and aCry5-like gene designated as Cry1I.

These genes can be grouped into three major families: i) Cry1 familygenes encode for proteins toxic to lepidopteran insects only, Cry1Aa,Cry1Ab, Cry1Ac, Cry1C, Cry1D, ii) Cry2 genes encode proteins toxic tolepidopteran and dipteran insects, Cry2A and Cry2B, and iii) Cry1B andCry5 genes encodes proteins toxic against lepidopterous and coleopteransinsects. A pool of at least nine genes were present in this singlebacterium, which covers the widest range of insecticidal activityrecorded in the scientific literature.

Identification of Additional Genes with Potential Insecticidal Activity

In the same manner as the above example, additional genes werediscovered to exist in BtC18 which are potentially responsible forgiving this strain its broad spectrum of insecticidal activity. Cry1F,Cry1G, Cry1K, and Cry1M were found as well as nematode specific genes,designated nem1, nem3, nem5 and nem7. In addition, a vegetative toxinwas discovered in BtC18 which gives a PCR generated band of the sizeexpected for strain BtHD1. This vegetative toxin is produced duringvegetative growth and kills black cut worm of corn (BCW).

Cloning of Genes

All the genes detected by PCR in BtC-18, mentioned above, were clonedinto BlueScript sk+II plasmid vector. Custom made DNA primers againstthe toxin domains of the respective genes were used in PCR to amplifyand target the N-terminus part of the genes. The amplification productswere eluted from the gel, biocleaned and ligated to blunt endedpBlueScript. E. coli was subsequently transformed with this vector, and,recombinants containing the target genes were identified. The positiverecombinants were determined using the specific DNA primers and PCR toidentify the expected gene products. Recombinants clones were expressedin E. coli and the total cellular proteins of the recombinants wereanalyzed by SDS-PAGE. For better expression, the genes were cloned intoan expression plasmid pTrc99.

Cry1I Gene

Analysis of the Cry1I expression products by SDS-PAGE was performed. Twoproteins were produced, a 70 kDa protein and a smaller 58 to 60 kDaprotein which are not produced by E. coli transformed with pTrc99 alone.The Cry1I gene was subcloned for the purpose of restriction mapping andDNA sequencing. Three nucleotide sequences of three different segmentsof the Cry1I gene from BtC-18 were obtained using the dideoxy chaintermination method (sequences not shown). The three segments were the 5′and 3′ ends of the gene as well as a middle segment. The nucleotidesequence of these three segments of the Cry1I gene from BtC-18 werecompared using Blast server to nucleotide sequences of the publishedsequences of several different Cry5 genes. By this method identities andhomologies of the nucleotide and amino acid sequences were determined.The closest relative to the C18 Cry1I gene is that of entomocidus. C18Cry1I shares 97% nucleotide identity and 92% amino acid identity withthe entomodidus Cry5. The complete nucleotide sequence of the C18 Cry1Ihas been obtained and is represented in FIG. 5.

Cry1I Activity Against Rootworm

The C18 Cry1I gene was inserted into the expression vector pTrc99 withIPTG induction. E.coli strain BL21 was transformed with vector plusCry1I insert and empty vector as a negative control. Once the bacteriahad grown to the appropriate cell density, they were pelleted andresuspended in 11 ml of buffer (10 mM MgSO4, 0.3% Tween-20, 2 mM PMSF).The cellular suspension was sonicated and protein extracted. Theconcentration of the protein extract was 45 mg/ml. WCRW (Western CornRootworm) larvae were challenged with various concentrations of theprotein extract and mortality scored after three days. Results of thisassay demonstrates a dramatic increase in WCRW mortality compared to thenegative control at doses as low as 2.3 μg/cm². At these levelsmortality of the larvae exposed to the extract containing Cry1Ip is up55% compared to the mortality of the larvae unexposed. At 11.8 μg/cm²the mortality is up 80%. See Table 3. This is clear evidence that Cry1Iis one of the constituents in the BtC18 insecticidal arsenal thatdemonstrates anti-rootworm activity.

TABLE 3 Est. Cry1I dose Extract vol. Dead (WCRW) Live (WCRW) % Mortality(μg/cm²) 0.1 ml 6 2  75 2.3 0.5 ml 8 0 100 11.8 1.0 ml 8 0 100 22.3 2.0ml 8 0 100 44.7 Control 3 12   20 0

Purification of Insecticidal Proteins from BtC-18 FPLC Separation ofBtC-18 crystal proteins

Protein fractions from BtC-18 were dialyzed, solubilized and thenpurified on a Mono Q column following the FPLC procedure recommended bythe manufacturer:

1. Portions of the fractions were loaded onto the column and elutedusing NaCl gradients.

2. Each individual fraction was bioassayed against different insects,analyzed by SDS-PAGE, and ligand binding studies were performed usingthe midguts of different insects to identify specific receptor proteins.

Isolation of Crystal Proteins from BtC-18.

The bacterium BtC-18 was cultured in liquid sporulation medium(T3-medium) at 30°-32° C. and the crystal proteins were purifiedaccording to Lee et al. (1985) Biochem. Biophys. Res. Commun.,126:953-960 using the following procedure:

1. The cells were grown to the spore stage. The crystal proteins werereleased into the medium following autolysis of the cells.

2. The spore-crystal complexes were separated by centrifugation (10,000rpm, for 10 min at 4 C). The complex was washed twice each with 1Mpotassium chloride in deionized water.

3. The pellet was resuspended in distilled water and homogenized with aDounce homogenizer followed by centrifugation to separate the proteincomplexes. This step was repeated two times.

4. The pellet (2-4 g) was suspended in a 68% Renografin gradient (9.6 mlof 2% Triton X-100 and 20.4 ml of Renografin). The mixture washomogenized and subjected to sonication 10 times (20 pulses each time).

5. The mixture was centrifugated at 27,000 rpm for 15 hours. The viscoustop band on the gradient contained the crystal proteins. The purity ofthe crystal proteins was checked by microscopic examination.

6. The crystal pellet was resuspended in 30 ml of 55% Renografin, 1%Triton X-100, homogenized with a glass Dounce homogenizer and sonicated10 times as above.

7. The mixture was centrifugated again at 27,000 rpm for 15 h and thecrystal proteins were precipitated as a white pellet.

8. The supernatant was carefully removed and the pellet was washed threetimes with distilled water before lyophilization.

Solubilization of the Crystal Proteins from BtC-18

Solubilization of the crystal proteins was accomplished according to themethods of Dai et al. (1993) Insect Biochem. Mol. Biol., 23:273-283 andHöfte et al. (1986) Eur. J. Biochem., 161:273-280.

1.50 μg of the purified crystal proteins were dissolved in 5 ml of thefollowing solution:

Sodium carbonate (pH 9.5) 50 mM Dithiothreitol (DTT) 10 mMPhenylmethylsulfonyl Fluoride (PMSF)  5 mM Ethylenediaminetetraacetate,disodium 10 mM

The mixture was incubated at 37° C. for 80 min. with continuous shaking.

2. The mixture was spun at 14 K rpm for 15 min. and the supernatant wasanalyzed by SDS-PAGE.

3. The pellet was dissolved in the following solution:

Sodium hydroxide (pH12) 50 mM Dithiothreitol (DTT) 10 mMPhenylmethylsulfonyl fluoride (PMS)  5 mM

and incubated at 37 C/1 hour.

4. The supernatant was collected again by centrifugation at 14 k for 15min.

5. The fractions were dialyzed against Tris-HCl (50 mM, pH9) containing50 mM NaCl.

After purification, the proteins are assayed for activity againstinsects of interest.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A biologically pure culture of a Bacillusthuringiensis strain having pesticidal activity against nematodes andagainst insects from the orders Lepidoptera, Diptera and Coleoptera,wherein said strain is BtC-18 deposited as ATCC Accession No. 55922, ora mutant thereof.
 2. A pesticidal composition comprising a Bacillusthuringiensis strain having pesticidal activity against nematodes andagainst insects from the orders Lepidoptera, Diptera and Coleoptera,wherein said strain is BtC-18 deposited as ATCC Accession No. 55922, ora mutant thereof.
 3. The pesticidal composition of claim 2, furthercomprising at least one member selected from the group consisting ofcarriers, surfactants, adjuvants, fertilizers, micronutrient donors,herbicides, insecticides, fungicides, bacteriocides, nematocides, andmollusocides.
 4. The pesticidal composition of claim 2, wherein saidpesticidal activity comprises activity against rootworm.
 5. A method forkilling pests comprising applying a pesticidal composition to theenvironment of at least one target pest, said pesticidal compositioncomprising a Bacillus thuringiensis strain having pesticidal activityagainst nematodes and against insects from the orders Lepidoptera,Diptera and Coleoptera, wherein said strain is BtC-18 deposited as ATCCAccession No. 55922, or a mutant thereof.
 6. The method of claim 5,wherein said pesticidal activity comprises activity against rootworm. 7.The method of claim 5, wherein said pesticidal composition furthercomprises at least one member selected from the group consisting ofcarriers, surfactants, adjuvants, fertilizers, micronutrient donors,herbicides, insecticides, fungicides, bacteriocides, nematocides, andmollusocides.
 8. The method of claim 5, wherein said applying is by anapplication method selected from the group consisting of leafapplication, seed coating, and soil application.